Regulation of Nitrogen Metabolism and Gene Expression in

25 Pages·2004·4.52 MB·English

MICROBIOLOGICAL RzvIEws, Sept. 1981,p.437-461 Vol.45,No.3
0146-0749/81/030437-25$02.00/O
Regulation of Nitrogen Metabolism and Gene Expression in
Fungi
GEORGE A. MARZLUF
Depalment.ofBiochemistryandProgra inMolecular, CellularandDevelopmentalBiology, TheOhio
State University, Columbus Ohio 43210
ITRO04DUCTON 437
.............................................................
NITREAT...E..............................E.D..........E...438
NiRATE REDUCTASE ANDAUTOGENOUS CONTROL .. 440 D
TURNOVEROFNiREATEREDUCTABSE.44...1..... o
w
PURINlNCEATABO-LISM..................4.............................441
n
PRO11UN4ECATABOLISML ..........446.....................4.4.6 lo
ACETAMEDEUTILIZATIONANDINTEGRATOR GENE .448 a
NrIROGENCATABOLIE REPRESSIONN: GENgI'IC STUDIE 450 d
PATHWAY-SPECIFIC CONTROL . ............... 451 e
d
REGULATORYRECOGNIMIONSEQUENCES? ............................... 451
f
GLUTAMATEDEHDROGENASE ........................................... 452 ro
GLUTAMATE DMElHYDROGENASE: A REGULATORY PROTEIN? 453 m
..........
GLUTAMIMMNE .E N.T.H.A.S3E 454
h
MERCHANISRMOF NIROGENCATABOIXTE REPRESSION ............. 455 t
t
CLOSING SANDFUTUREPROSPECTS ........................... 457 p
LITERATURE,CCYIRTEED 458 :/
. /
m
INTRODUCTION quires that two conditions be met. First, there m
b
mustbealiftingofnitrogencataboliterepression r
Nitrogen isamajorelementfoundinmanyof (also called amnium repression); second, in .a
the simple compounds and nearly al of the manycases,specificinductionoftheenzymesof sm
complexmacromoleculesoflvingcells. Proteins aparticularcatabolhcpathwaybyasubstrateor .
andnuclei acidsareespially richinnitrogen. intermediateofthepathwaymustalso occur. A or
Thus,itshouldnotbe is at asubstan- major goal is to gain an understanding of the g/
tialcellularinvestmentismadeinthemetaboeic genetic and metabolic signals that areresponsi- o
machinery compising nitrogen catooic path- ble for the separate steps ofrepression and in- n
ways to ensure a constant nitrogen supply for duction. A
p
growth. Extensive studies ofnibtrgen metabo- Themajorityoftheavailableinformationand r
las ansd its, control have been carried out in evidence concerning the regulation ofnitrogen il
1
three fungi, Neurospora crassa, Aspergilus metabolism in the above fngi is derived from 0
nidzdns, and Saccharomyces cerevisiue. In geneticanalysisofvariousstucturalandcontrol ,
2
eachof'these major control systems mutants Thus, it is appropriate to summarize 0
exist to regulate nitrogen metabolism. In this certainconceptsrelevanttosuchgeneticstudies. 19
review, certainnitrogencataboicpathwayswill Genetic evidence alone is usually (perhaps al-
b
bebrieflydescnrbed, andtheregulatorymecha- ways)ifficienttoestablishthelevelatwhich y
nisms which control nitrogen mebotabism in theproduct ofaregulatory geneacts; infact, it g
thesefungiwil be onsidered indepth.Anumn- iseven difficut tounequivocallyidentifyareg- ue
berof_eeletreviewsconceningnitrogenme- ulatory gene with only genetic evidence, since s
tbolism andvariou relatedtopic infungi are other types of mutants can yield a phenotype t
available (23, 29, 30, 59, 60, 65, 66, 84, 88, 99). similarto that expected for mutants ofcontrol
Although certain compounds, partclarly am- genes.Tleabilitytoobtaindifferentmutantsof
monia, glutamate, and gluine, are favored thesame genewhichyieldoppositephenotypes
nitrogen sources, thesefungiarecapable ofuti- (constitutiveversusnull)providesatleaststrong
manydiversesecondarysources,including indirect evidence for a true regulatory gene.
nitrate nitrite, purins, proteins, numerous However, theinabilitytofindmutantswithop-
ramino acids, acetamide, and even acrylamide. positephenotypesdoesnotexcludearegulatory
The use of these secondary nitrogen somrces roleforaparticulargene.
invariably requires the synthesis of catabolic Most ofthe regulatory genesofthe fumgi are
enzymesor, msomecases, anactivationofpre- not linked to their structural genes, although
viously existing enymes. De novo synthesis of certainexceptionstothisruleexist.Aregulatory
many of the nitrogen-regulated enzymes re- mutant has two possible phenotypes: it may
437
438 MARZLUF MICROBIOL. REV.
cause the constitutive expression (partial or inatwo-electrontransferreaction;nitriteisthen
complete) of the relevant enzymes, or it may converted to ammonium in a complex reaction
leadto the absence (orreduced levels) ofthese involving the transfer of six electrons. Nitrate
sameenzymes(nullmutant).Itisdiagnosticthat reductase ofNeurospora isadimeric enzyme of
mutation within positive-acting control genes molecular weight 228,000 and is composed of
willfrequentlygiverisetonullmutantsandonly two identicalpolypeptide subunits (11). Nitrate
rarely yield constitutive mutants. Conversely, reductase possesses a molybdenum-containing
mutants of negative-acting control genes are cofactor, which is also found in other molybde-
usually constitutive, and only infrequently are num enzymes, including xanthine dehydroge-
null phenotypes obtained. We will encounter nase (XDH) (84).Nitritereductasealsoappears
examplesofbothpositiveandnegativenitrogen tobe ahomodimer ofmolecularweight 290,000
control genes in the fungi. Another important and contains an unusual siroheme prosthetic D
feature isthe presence of"major" and "minor" group. The synthesis ofboth nitrate reductase ow
control genes; the major nitrogen control genes and nitrite reductase requires induction (by ni- n
integrate the expression of numerous enzymes trate) and is completely repressed by ammo- lo
ofnitrogenmetabolism,whereastheminorcon- nium. ad
trol genes are pathway specific and affect only Mutants in alarge number ofunlinked genes e
the enzymes ofa particular catabolic pathway. inNeurosporaleadtotheinabilitytousenitrate d
Finally, we can anticipate identification ofcon- as a nitrogen source (Table 1). The nit-3 gene fro
trolregionsadjacent to structuralgenes (which apparently encodes the major polypeptide of m
may serve as recognition sites for regulatory nitratereductaseandisunlinkedtonit-6,which h
proteins) as cis-acting mutants, which yield specifies nitrite reductase (94). By contrast, tt
eitheraconstitutiveoranullphenotype.Aswill niaDandniiA,whichencodenitrateandnitrite p:
be seen, these types ofmutants can be particu- reductase, respectively, in Aspergillus, are //m
larly difficult to define with genetic evidence tightlylinked (93). InAspergillus, anadditional
m
alone. fiveorsixloci,knownasthecnxgenes,together b
It is occasionally necessary to be reminded specify a molybdenum-containing cofactor r
.
that changes in the amount ofenzyme activity shared by nitrate reductase and XDH I and II a
s
observedinmutantsandinvariousstrainsgrown (Table1).Thiscofactorconsistsofapolypeptide m
under different nutrient conditions can result component encoded by cnxH and a molybde- .o
from effects atmany differentlevels ofcontrol. numligandgroupwhoseformationisapparently rg
These include transcription (and possibly proc- instructed by the other cnx genes (23, 84). A /

essing and secretion of messenger ribonucleic similar situation exists in Neurospora; at least on
acid[mRNA]),translation,mRNAstability,en- four genes, nit-i, nit-7, nit-8, and nit-9, seem
A
zyme maturation (from an inactive precursor), homologous to the Aspergillus cnx genes and p
leonwz-yomrehitguhr-nmoovlere,culaanrd-weeingzhytmienhiibnihtiobrist)i.onSin(cbey amroelryebsdpoennsuibmlecofofracstyonrth(e9s4i)s.aTnhdeasnsiet-m9bllyocoufsthies ril 1
many studies of nitrogen regulation in fungi complex and contains three complementation 0,
involve measurement of enzyme activities in 2
crude extracts, it isvirtually impossible such TABLE 1. GenesinNeurosporaandinAspergillus 0
cases to know which of the above steipns are whichaffectthenitrateassimilatorypathway 19
implicated. Accordingly, an attempt will be Geneticlocus b
made to weigh the strength ofevidence as itis Function Neuro y
presented andtodiscussanyavailableinforma- spora Aspoergillus gu
tionwhichimpliescontrolatcertainlevels, e.g., e
de novo enzyme synthesis or mRNA synthesis. Structuralgenefornitrate nit-3 niaD st
Finally, the cellular location of enzymes, sub- reductase
strates, effectors, and regulatory components is Structuralgenefornitrite nit-6 niiA
reductase
of paramount importance to regulation as it Geneswhichspecifya nit-i cnxABC
occurs in vivo; however, since this itself is a molybdenumcofactor nit-7 cnxE
comprehensive area in which excellent reviews nit-8 cnxF
are available (28-30), no attempt will be made nit-9 cnxG
to coverthissubjecthere. cnxH
Pathway-specificcontrol nit-4 nirA
NITRATE REDUCTASE gene (mediatesinduction)
Majornitrogenregulatory nit-2 areA
Inorganic nitrate serves asanexcellentnitro- genes (mediatesnitrogen tamA
gen source for both Neurospora and Aspergil- cataboliterepression)
lus.Nitrateisenzymaticallyconvertedtonitrite
VOL. 45 N METABOLISM AND GENE EXPRESSION IN FUNGI 439
groups, indicating that it may comprise three Thetightlinkage ofniiA andniaDobviously
closely linked genes, similar to the complex brings up the question ofwhether they may be
cnxABClocusofAspergillus. regulatedasaunitinanoperontypeofarrange-
In addition to the genes encoding structural ment. Both genesrequire the product ofnirA+,
componentsofthenitrateassimilatoryenzymes, the pathway-specific positive control gene, and
at least two types of regulatory mutants are ofareA,apositive-actingmajornitrogencontrol
found in Neurospora (and Aspergillus) which gene, for expression. However, similar control
cannotusenitrateornitriteforgrowthandlack genesregulatetheanalogousbutunlinkedstruc-
both nitrate and nitrite reductase. One control tural genes in Neurospora; moreover, synthesis
gene ofNeurospora, nit-4, is pathway specific, ofnitrateandnitritereductaseinAspergillusis
and mutants of it lack nitrate reductase and not strictly coordinately regulated, and cis-act-
nitrite reductase but do not affect any other ingmutantswhichmaydefinepromoterorcon- D
nitrogen-related enzymes. All mutants of nit-4 trol elements are found which are specific for o
w
are null, which implies that this gene is a posi- justoneortheotherofthesegenes. Inaddition,
n
tive-actingregulatorylocus.Althoughoriginally all nirAc mutants studied to date lead to much lo
another pathway-specific control gene, desig- higher constitutive expression ofnitrate reduc- a
d
natednit-5, wasreported inNeurospora, itwas tasethannitritereductase (9);thisresultismost e
recently demonstrated nit-4 and nit-5 are ac- easilyinterpretedtosuggestthatniiAandniaD d

tuallyrepresentativesofasinglelocus,hereafter eachpossessarecognitionsiteforthenirAgene fr
referred to as nit-4 (94). This control gene is product,andthattheydiffersufficientlytohave o
m
apparently responsible for mediation ofthe in- markedlydifferentaffinitiesforthealteredprod-

duction ofnitrate and nitrite reductase; it has uctspecifiedbythenirAc mutant. h
t
been postulated that the nit-43 gene product is Arstetal. (9)havecompletedadetailedstudy tp
a regulatory protein which, upon binding the ofnis-5, which behaves as a tightly linked cis- :/
/
inducer nitrate, binds at recognition sequences acting constitutive mutant that controls niiA m
adjacent to the nit-3 andnit-6structural genes expression. Thenis-5mutantpermits about8% m
to "turn on" theirexpression (59, 94). A second constitutive expression ofjustnitritereductase, b
r
positive-acting regulatory gene of Neurospora, which isindependent ofanynirA gene product .a
designatednit-2, controls nitrate andnitrite re- andisnotammoniumrepressible norinducible; s
m
ductasealongwithmanyothernitrogen-related upon induction, the nis-5 mutant strain pro-
.
enzymes and mediates nitrogen catabolite ducesabout40%ofthewild-typelevelofnitrite o
r
repression (36, 59, 78). reductase (77). Itwas recently determined that g
/
In Aspergillus, a pathway-specific control the nis-5 phenotype actually resulted from a o
gene (nirA) for induction and a major control nonreciprocal translocation in which asegment n

gene (areA) whichmediatesnitrogen catabolite of chromosome II was inserted between the A
p
repression appear to be analogous to the Neu- niaA and niiA genes (9). It was suggested that r
rospora regulatory genes described above. The the inserted segment contains a low-level pro- il
1
fact that constitutive nirAc mutants do not re- moterwhichisresponsibleforthelowconstitu- 0
quireinductionbutstillarenitrogenrepressible tive expression ofniiA and that a natural pro- ,
2
fornitrate reductase, whereas constitutive-type moter specific for niiA, which resides between 0
areA mutants (areAd) still require induction niaD and niiA, was still present in the translo- 1
9
(butarenonrepressible),supportsthesuggested cation mutant and was responsible for the in-
b
rolespostulated forthese controlgenes. ducible expression. The reduced expression y
The structural genes which encode nitrate upon induction could be explained by the pres- g
reductase (niaD) and nitrite reductase (niiA) ence of neighboring chromosome II sequences u
e
areverycloselylinkedandprobablycontiguous which place the regular promoter in a new en- s
t
in Aspergillus. Tomsett and Cove (93) have vironment. It perhaps deserves emphasis that
accomplished a detailed genetic analysis ofthe the constitutive phenotype of nis-5 suggested
niaD-niiA region by deletion mapping. They that itwas amutant ofa control gene when, in
found that approximately 1% of spontaneous fact, itresultedfromatranslocation. The avail-
chlorate-resistant mutants behaved as niaD- ableinformationisconsistentwiththeexistence
niiA double mutants, which in every case were oftwo such different promoters for niiA in the
shown to be deletions. The deletion mapping nis-5strain, butthe evidenceisnotcompelling.
confirmed the extremely tight linkage of these Itdoesseemclearthatseparatepromotersexist
twogenesand,moreover,permittedlocalization fortheniaDandtheniiAgenes,whichsuggests
ofa cis-acting regulatory mutant which affects thattheselinkedgenesarecontrolledseparately
niiA geneexpression withintheregionbetween although in a parallel manner. Arst et al. (9)
niiA andniaD (93). speculated that the niiA gene is normally ex-
440 VOL. 45 N METABOLISM AND GENE EXPRESSION IN FUNGI
pressedintwoways,firstasabi-orpolycistronic tasesynthesizedwillbeinactive (72).Thepadis
mRNA which initiates at the niaD gene and subsequentlymovedtotranslationmediumcon-
reads through into niiA sequences, and second taining glutamine (to prevent any additional
by transcription of niiA as a monocistronic mRNA synthesis) plus Mo, so that any accu-
mRNA from a promoter lying between niaD mulated mRNA can then be translated into
and niiA. Evidence necessary to establish this activenitratereductase. Culturesareharvested
possibility should include a demonstration that andassayedfortheircontentofnitratereductase
niiA codinginformationoccursontranscriptsof activity, with the assumption that the capacity
radicallydifferentsizes.However,sinceonlythe to synthesize the enzyme is equivalent to the
first coding sequence in a polycistronic mRNA cellular content of mRNA (72). This approach
can apparently be translated in eucaryotic sys- permitted several tentative conclusions. Gluta-
tems (70),itseemsprobablethatniiAandniaD mine prevents the accumulation of nitrate re- D
are transcribed separately, each from its own ductasemRNAbutdoesnotaffectmRNAtrans- o
w
promoter-controlregion. lationnorenzymeactivity,indicatingthatnitro-
n
Garrett and his colleagues have presented gen repression occurs at transcription. It is im- lo
convincing evidence that induction of nitrate portant to note that presumptive mRNA accu- a
reductase in Neurospora involves de novo en- mulated under inducing conditions, and that d
e
zymesynthesis.Whennoninducedcultureswere bothnitrogenderepressionandnitrateinduction d
transferred to medium containing 90% deute- wererequiredformRNAaccumulation (73).In- f
r
rium oxide, induction by nitrate yielded nitrate ducedcultureswhichcontainsuchaccumulated o
m
reductase ofuniformly heavydensity (11). This mRNA begin synthesizing active nitrate reduc-

result arguesfor de novo enzyme synthesis and tase immediately upon transfer to translation h
t
rulesoutthepossibilitythataninactiveprecur- medium, whereas uninduced cultures display a tp
sor or even any major component of nitrate 20-minlagbeforetheonsetofnitratereductase :/
/
reductase existed before induction. Further- synthesis; thisdifferencealsoarguesthattrans- m
more, Amy and Garrett (1) usedhighlyspecific latablemRNAcanaccumulateunderconditions m
immunoelectrophoretic techniquestodetectni- in which active nitrate reductase cannot be b
r
trate reductase protein, even partial chains, in- made.Theresultsstronglyarguethatcontrolof .a
dependent ofany enzymatic activity. The anti- nitratereductase,atleastinNeurospora,occurs s
m
bodies used inhibited all known activities of atthetranscriptionallevelandthatbothinduc-
.
nitratereductase anddidnotdisplay anycross- tion and nitrogen catabolite repression control o
r
reaction with XDH or nitrite reductase. They thisstep,oracloselyrelatedone,suchasmRNA g
/
found that uninduced wild-type cultures which processing or transport. If any control of this o
lack nitrate reductase, as well as nitrogen-re- enzymeoccursatthetranslationalorpost-trans- n

pressed cultures, lack any cross-reacting mate- lationallevel itapparently makesonlyaminor A
p
rial (CRM). Moreover, nit-i, nit-3, and nit-6 contributiontothe overallregulation. r
mutants contain CRM when induced, as ex- il
NITRATE REDUCTASE AND 1
pected,butthetworegulatorymutantsnit-2and 0
nit-4lackanydetectableCRM(1).Theseresults AUTOGENOUS CONTROL ,
2
convincinglydemonstratethatnitratereductase InAspergillus nidulans, synthesis ofnitrate 0
is not controlled by activation ofa preexisting reductase and nitrite reductase is controlled 1
9
precursor protein orbysomeformofinhibition both by nitrate induction and by ammonim
b
ofa cryptic enzyme, but involves de novo syn- repression. Furthermore, nitrate reductase has y
thesis. been postulated to play an autogenous regula- g
Premakumar et al. (72) have used a novel toryrole, controllingitsownsynthesisandthat u
e
approach tostudythesynthesisandstabilityof ofnitrite reductase. Indeed, Cove andPateman s
Neurospora nitrate reductase mRNA. Tung- (24), who suggested such a control function for t
sten, a molybdenum analog, inhibits the devel- nitrate reductase, may have been the first to
opment ofnitrate reductase activity because it propose autogenous gene regulation. The key
is actually incorporated into nitrate reductase observationthatsuggeststhishypothesisisthat
(in place of Mo), yielding an inactive enzyme. manymutants whichlacknitrate reductase ac-
This feature permits experimental approaches tivityneverthelessdisplayconstitutivesynthesis
in which nitrate reductase mRNA can be accu- of nitrite reductase and of nitrate reductase
mulatedintheabsenceofsynthesisofanyactive CRM. Cove (23) found that 17 niaD mutants,
enzyme. Uninduced mycelial pads are trans- including onedeletedforalargesegmentofthe
ferredto"transcriptionmedium"whichcontains niaDgene (outofatotalof27examined), pro-
nitrate as the inducer plus tungsten, so that duced nitrite reductase and nitrate reductase
mRNA can accumulate but any nitrate reduc- CRMconstitutively. Moreover,certaincnxmu-
MARZLUF MICROBIOL. REV. 441
tants also resulted in the constitutive synthesis TURNOVER OF NITRATE REDUCTASE
of nitrite reductase. Thus, it appears that the
Attention has been given to the inactivation
nitrate reductase protein, probably in atleast a
ofnitrate reductase, which occurs both in vivo
near-native conformation, isneeded fornormal
andinvitro, andthepossibilitythatturnoverof
regulation.Theresultsindicatethatsynthesisof this enzyme could have a regulatory function
the nitratepathway enzymesnormallyrequires
(91, 100). In Neurospora, two different decay
nitrate induction, but that when nitrate reduc- mechanismsfornitrate reductase canbe distin-
tase is missing, their synthesis still requires a guished in vivo. One decay system, designated
nirA+ protein but not nitrate. Thus, nitrate re- "N," is especially prevalent in nitrogen-starved
ductase may itself provide the nitrate recogni- cells, is very sensitive to ethylenediaminetetra-
tion site, whereas the nirA+ product may be acetic acid and cycloheximide, and decreases D
responsible for the specific recognition of the with mycelial age. The second system, termed o
structuralgenes.Asimpleinteractionsuggested "A,"isrelativelyinsensitivetoethylenediamine- w
bitysetlhfebsiendcsontsoidtehreatniiornAs+isprtohtaetinniwtrhaetenrneidturcatteasies tweittrhaamceytciecliaaclidaagned. cTyhcleohNexismyisdteeamnfdoirncnrietarsaetse nloa
absent,therebyleadingtoaconformationofthe reductasedecayisapparentlyageneralturnover d
nirA+ product which is unable to initiate niiA system which appears during nitrogen starva- ed
andniaDexpression. Upon bindingnitrate, the tion. The post-translational effect of nitrate f
reductase maynolongerbindto thenirA+ pro- probablyresultsfromitsstabilizationandpartial ro
tein (or its conformation changes) so that the protection ofthe enzyme fromsystem N. m
nirAproteincanturnonexpressionofthestruc- The existence oftwo systemsforinactivation h
tural genes. Accordingtothissuggestion, amu- ofnitratereductasehasalsobeendemonstrated ttp
tant completely lacking nitrate reductase, or in vitro (91). Inactivator I was present in all :/
possessinganaltered form incapable ofbinding mycelia tested, regardless of nitrogen growth /m
the nirA protein, should no longer require ni- conditions,andwaspresentinthenit-2mutant; m
trateinductionforsynthesisofnitritereductase it may correspond to system A. Inactivator I is b
(ornitratereductaseCRMifrelevant).Another inhibited by a specific thermostable inhibitor r.a
suggestedmechanismisthatnitratereductaseis presentin boiled crude extracts ofNeurospora. s
a repressor and directly binds to deoxyribonu- Inactivator II, also studied in vitro, is nitrogen m
cleic acid (DNA) recognition sequences to re- repressible and is missing in nit-2mutants; it is .o
press structural gene expression unless the in- phenylmethylsulfonyl fluoride sensitive and rg
ducernitrate is present (84). In thisview, how- very active at pH 5. This inactivator is appar- o/
ever, it is not clear what function the nirA ently a serine protease and may be the one n
proteinwouldhave. responsible for rapid turnover ofnitrate reduc- A
Althoughtheautoregulatoryroleproposedfor taseinyoung,nitrogen-starvedcells(systemN). p
nitrate reductase stems entirely from genetic InactivatorIIisalsofoundinassociationwitha ril
evidence, and thus must remain speculative, it 1
specific thermostable inhibitor. The inhibitors 0
seems very important that future work be di- areexcludedbySephadexG-25,andbothappear ,
rectedatprovidingadditionalevidenceforsuch 2
tobesmall,thermostableproteins (91).Thetwo 0
afunction andelucidating themolecularmech- inactivators are separable on Sephadex G-150, 1
anism for autogenous control. Although many 9
and both have properties which indicate that
possible models canbesuggested, each ofthem b
they are proteases. The results presently avail- y
should permit specific predictions for in vitro ablesuggestthatneitherofthesedecaysystems g
experimentation. Thus, the first model sug- isspecificfornitrate reductase; theybothprob- u
gested above predicts that nitrate reductase e
ablyplayonlyageneralroleinproteinturnover s
should bindto (oralterinsome otherway) the ratherthananyspecial regulatory function. t
nirA+ product and, moreover, that the binding
should be abolished in the presence of N03.
PURINE CATABOLISM
This, in fact, could provide a direct means to
searchforthepostulatednirA+geneproteinvia Another area of nitrogen metabolism which
affinity chromatography techniques, since has been extensively studied in Aspergillus,
highly purified nitrate reductase is available. Neurospora, and yeasts is purine catabolism
One might also expect that nitrate reductase (Fig. 1). Although the pathway itself and its
might, in part, be localized within the nucleus regulation are very similar in Aspergillus and
for its regulatory fiuction. Alternatively the Neurospora (79, 86), striking differences are
nirA+ productcould be aDNA-bindingprotein presentinyeasts (21).
foundinthecytoplasm (complexedwithnitrate The conversion of hypoxanthine to xanthine
reductase) and able to enter the nucleus only and then to uric acid in Aspergillus has been
uponnitrate induction. studied in detail recently (89, 90), and several
442 MARZLUF MICROBIOL. REV.
>,! s"" ADENINE
NICOTINATE
hxn S
--Purine Dehydrogenosez HYPOXANTHINE
hxA
flr Purine Dehydrogenase I--i. .See GUANINE
6-HYDROXY %
f
NICOTINATE '
"~ XANTHINE
D
xa Al
o
XANTHINE w
ALTERNATIVE n
""----I ) PATHWAY lo
a
URIC ACID d
e
d
| ua Z fr
o
URICASE
m

h
ALLANTOIN tt
p
:
/
/
|alx m
allontoinase m
b
r
.
ALLANTOIC ACID a
s
m
.
aaX o
r
allantoinase g
/

o
ureidoglycollate -glyucroelildaos-es UREA n A
p
r
ureB il
1
urease 0
,

2
AMMONIA 0
1
FIG. 1. Pathway forpurine catabolism in Aspergillus. The structural genes which specify most ofthe 9
catabolic enzymes aregiven, butregulatorygenes areomitted. Neurosporapossesses an identicalpathway, b
y
exceptthatthexanthinealternativepathwayandpurinedehydrogenaseIIhavenotbeenexamined.Boththe
g
pathwayanditsregulationdiffersignificantlyinS. cerevisiae. u
e
s
earlier uncertainties are now largely resolved. hxSstructuralgene.Bothpurinedehydrogenase t
Aspergillus possesses two enzymes which cata- IandIIactivitiesaremissinginallcnxmutants,
lyze the hydroxylation of hypoxanthine (90). whichalsolacknitratereductase,indicatingthat
Purine dehydrogenaseI,thephysiologicallysig- allthreeofthese enzymesshareacommonmo-
nificantenzymeforpurinecatabolism, catalyzes lybdenum-containing cofactor. Moreover, pu-
theconversionofhypoxanthinetoxanthineand rine dehydrogenase I and II activities are both
ofxanthinetouricacid; thisenzymeisspecified missinginamutantdesignatedashxB,although
by the hxA gene and is induced bythe product CRM corresponding to each enzyme is present
uricacid. PurinedehydrogenaseIIisinducedby (90), suggesting that they further share some
6-hydroxynicotinicacid,theproductofitsactiv- common subunit or perhaps require a common
ity upon nicotinic acid, its primary substrate, post-translational modification specified by
but it also has strong activity toward hypoxan- hxB+.
thinebutnotxanthine (90); itisencodedbythe It is interesting that each of these purine
VOL. 45 N METABOLISM AND GENE EXPRESSION IN FUNGI 443
dehydrogenase enzymes requires induction by The uaY- phenotype derives from a loss of
theproductofitsphysiologicallyrelevantactiv- function; i.e., uaYdeletions have a null pheno-
ity.Suchproductinductionseemstooccurwhen type,furtherindicatingthatthewild-typeuaYr
the substrate is an essential metabolite but the gene actively turns on expression of the struc-
product is metabolically dispensible. The hxB tural gene set. In agreement, the catabolic en-
mutant, which lacks both purine dehydrogen- zymes are all synthesized in uarY/uaY- dip-
ases,isneverthelessstillcapableofmetabolizing loids, although the amount of uaY product is
xanthinetoproduceuricacid. Thisreactionhas apparentlynotsufficienttoevokefullexpression
been shown to take place by the "xanthine al- oftwosetsofstructuralgenes;thus,theenzyme
ternativepathway,"whichisabsentinamutant levels are slightly less than half that found in
designated xanAl (89). Since the alternative uaYr/uar diploids. It is particularly interest-
pathwayisactiveinallcnxmutants,itisobvious ing that the expression ofuaYr is nucleus lim- D
o
that no Mo-containing cofactor is required. As ited and that uaYr can turn on the expression w
one would predict, the double mutant hxB of uapA+ in the same, but not a different, nu- n
xanAl completely blocks all uric acid produc- cleus. This result is especially convincing since lo
tion. complementation occurred indiploidswhereall a
d
Eight structural genes ofAspergillus which genes were in the same nucleus but failed in e
encode thepurine catabolic enzymes are allun- heterokaryons in which uapA+ and uaYr were d
linked to one another, but are controlled as a inseparate nuclei (85). fro
group by the pathway-specific control gene One ofthe structural genes controlled bythe m
uarY,whichmediatesuricacidinductionofthis uaYr product, uapA+, encodes a uric acid-xan-
h
entire set of enzymes (86, 87). Uric acid is the thine permease. A tightly linked, cis-dominant tt
onlyphysiological inducerofpurine dehydroge- mutant, called uap-100, is constitutive for the p
:
naseI,adeninedeaminase,anduricase,although permeaseanddoesnotrequireinducer,although //m
the2-and9-thioanalogsofuricacidareeffective itisstillfully dependentuponafunctional uaY m
as gratuitous inducers (95). By contrast, the product (10). This constitutes good evidence b
other purine enzymes are induced by both uric that the uaY product controls the uapA gene r.
acidandallantoin.Allantoinaseandallantoicase and, byanalogy, obviously also the other struc- a
s
are inducible in thewild type byboth uric acid turalgenesofthissameset.Theuap-100mutant m
and allantoin (86). The fact that these two en- has at least three related effects: (i) it shows a .o
zymes are inducible by allantoin alone in uaY 2.5-fold "uppromoter" effectbeyondthatofthe rg
mutantssuggeststhatallantoininductionisme- wild-type level; (ii) it is strongly constitutive /
diated by another mechanism (29). This entire (62%) in the absence of inducer, and (iii) it o
n
setofenzymesisalsosubjecttonitrogencatab- respondstotheareA102mutantproductaswell A
olite repression, mediated by the areA control as the areA+ product (85). These properties p
gpeantehwianyA,spuerregiidlolgulsy.coTlhlaeselaastndtwuoreeansze,ymaepspeoafrthteo tihmeplbyintdhiatngthseitueaspf-o1r0b0omtuhtathteiounasYomperohdouwctalatenrds ril 1
be constitutive or only slightly inducible inAs- theareAproduct,suggestingthesebindingsites 0
,
pergillus (86) andinNeurospora (79). may partially overlap each other. The uap-100 2
A preliminary fine-structure map ofthe uaY mutant almost certainly alters a control recog- 01
controlgeneandtheadjacentrelatedoxpAgene nition site which serves the uapA+ structural 9
hasbeen constructed byScazzocchio etal. (87). gene. The uap-100alteration ofthe recognition b
Ten differentalleles including one deletionmu- siteapparentlyallowsbindingofadifferentcon- y
tantarenonleakyandcannotbeinducedforany formational form ofthe uaYprotein than that gu
ofthe enzymes tested (uricase, adenine deami- recognized by the wild-type recognition se- e
nase, and purine dehydrogenase), whereas two quence,whichpresumablycanbindonlyauarY st
other well-separated uaY alleles (uaY109 and product (whoseconformationhasbeenchanged
uaY10) are leaky and only slightly inducible afterbindinginducer). Anothermutant, oxpA5,
for purine dehydrogenase and adenine deami- defines a gene located adjacent to uaY, but is
nase but highly inducible for uricase. Since all believed tobe separate because it complements
12 mutants, including the 2 leaky ones, fail to all uaY mutants, including a deletion in uaY
displayintrageniccomplementationinanypair- (87). TheoxpA5mutantdisplaysoxypurinolre-
wise combination, the uaYproduct maynot be sistance and is also partially constitutive for
ahomomultimeric protein. adenine deaminase, purine hydrogenase I, and
NouaYv (constitutive) mutantshaveyetbeen uricase. It has been speculated (87) that the
found, and the frequency of mutation to the oxpA gene codes for a protein which somehow
uaY- null phenotype and reversion pattern are limits the flow of inducers into the nucleus;
compatible onlywith a positive mode ofaction. however,thismustberegardedonlyasaninter-
444 MARZLUF MICROBOL. IREV.
esting possibility, since the evidence for it is tectedandpermittedpurificationoftwobinding
virtuallynonexistent. proteins.Bothofthesebindingpeaksaremiing
Usingimmunoprecipitationofuricaseandpu- inmutants with a putative deletion intheuaaY
rinedehydrogenaseI,WintheretaL (103)estab- gene, whereas maY109, a leaky mutant, shows
lished that induction oftheseenzymes involves only one peak, which elutesatadifferentposi-
de novo protein synthesis and that theenzyme tionfrom that ofeither ofthe wild-typepeaks.
proteinincreasesmproportiontoenzymeactiv- This result suggests that one or both ofthese
ityduringinduction.Theyalsomeasuredpurie protein peaks may represnta aY-codedpro-
dehydrogenaseI-anduricase-specificmRNAby tein. However, other proteins in a total cell
invitrotranslationofRNAextractedfromwild- extractmightalsobeexpectedtohinduricacid.
type and mutant strains underdifferent condi- Twoobviouscandidates, uricaseandpurinede-
tions of induction. The subunits of uricase hydrogenase,donotcorrespondtoeitherofthe D
(32,000 daltons) and purine dehydrogenase I proteinpeaks detectedinthiswork. o
w
(150,000 daltons) were identified asproducts in Oneinterestingpossibilityisthatoneprotein n
thetranslationmixturebyimimunoprecipitation peak is the uaY product and that the second lo
andsubsequentsodiumdodecylsulfate-gelelec- peakfoundinthewildtypeisoneoftheproteins a
d
trophoresis Induction of these two enzymes, under uaY control. A uaY deletion would be e
bothundercontrolofthe maYgene, wasshown expected to lack both proteins, whereas other d

tooccuratthelevelofproductionoftranslatable uaYmutants might possessa qualitatively dif- fr
o
mRNA. ferent maYproduct (but should be ing the m
Urease,thelastenzymeofthepurinecatabolic protein,controlledbytheaaYgene).Phil4ppides
h
pathway in Neurospora and AspergillU, is a and Scazzocchio (70) have purified sufficient t
t
constitutive enzyme, which is perhaps not sur- amounts of the major protein, which they be- p
:
prising since urea is also derived from arginine lievetobetheuaYproduct,tostudyitsbinding //
m
metabolism. Mutations atfourgenetic locipre- of the three inducers by equilibrium dialysis.
m
ventthe utlization ofureaas anitrogensource Theseresultsareprovocativeandmayrepresent
b
(58); ureA specifies a urea permease, whereas afirstinstance ofidentification ofagenetically r
.
mutantsoftheotherthree, ureB,-C,and-D,all definedregulatory geneproductinaeucaryote. a
s
lack urease activity. The ureB locus is thought However,considerableworkremainstobecom- m
tobethestructuralgene,andalthoughthefunc- pleted, first to demonstrate unequivocally that .
o
tion of wueC is unknown, it is interesting that themajorbindingproteinisindeedencoded by r
g
ureDapparentlyhasaroleintheproductionor uaY, and then to characterize its properties /

assemblyofanickelcofactorrequiredforurease andregulatorybehavior. o
n
activity.AdditionofnickelionstoureDrestored The pathway of purine catabolism and its
A
both urease activity and the ability to utilize regulation in Neuropora appears to be very
p
urea (58). similartothatjustdescribedinA pegius.No r
Considerable evidence was presented above pathway-specific gene analogous to the waY il 1
that the uaYr gene ofAspergillus encodes a gene ofAspergills has yet been identified in 0
positive-acting regulatory product which me- Neurospora.Structuralgenemutationsidentify- , 2
diatesuricacidinductionandturnsonthetran- ingthemajorenzymeshavebeenfoundandare 0
scription of at least eight unlinked structural unlinked; they are controlled both by uric acid 19
genes (86, 87). The cis-acting uap-100 mutant induction and nitrogen repression (59, 78, 79). b
(describedabove) isbelievedtodefineaspecific Synthesisofatransportsystemforuricacidand y

DNA recognition sequence for the uaY prod- xanthine is subject to nitrogen repression but g
u
uct. Uric acid and its 2- and 9-thio analogs are does not require uric acid induction (95). A e
inducers and thus should all bind to the maY separate hyponthine-adeniguanne per- s
t
productiftheproposedmechanismforitsaction mease is not regulated by either induction or
iscorrect.PhilippidesandScazzocchio(70)have repression (95). Induction ofurcaseapparely
attemptedtoidentifytheuaYproductbydirect occursattheleveloftranscription,sinceuricase-
examination ofwhole cellextractsforthe pres- specificmRNAcouldbeaccumulatedunderin-
ence ofa protein with the predicted character- ducing conditions in the presnce of cyclohex-
istics, namely, an ability to bind both inducer imide and subsequently trnslated to yield ac-
andDNA.Inoneapproach,inducer-bindingpro- tive enzyme (101). Two purine catabolic en-
teins were separated byphosphocellulose chro- zymes ofNeurospora have recently beenpuri-
matography and then identified by binding to fied and partially characterized; Lyon and Gar-
["C]2-thiouric acid. Inasecondtechnique, pro- rett (57) used a powerful immunoabsorption
teinswerefirstresolvedbyDNA-cellulosechro- technique to purify XDH, which was found to
matographyandthenidentified bytheirability be a dimeric enzyme composed of subunits of
to bind "C-labeled uric acid. Both methods de- molecular weight 155,000. Using imunoelectro-
VOL. 45 N METABOLISM AND GENE EXPRESSION IN FUNGI 445
phoresistodetectXDH,theydemonstratedthat way,inducesthesynthesisofallantoinase,allan-
the induced increase in XDH activity resulted toicase, urea carboxylase, and allophanate
from an equivalent increase in enzyme protein hydrolase. Another compound, oxalurate, acts
(57), indicating thatinduction involves de novo as a non-metabolizable inducer of these same
enzyme synthesis. Wang and Marzluf (102) re- enzymes. All ofthese enzymesarealsonitrogen
cently purified Neurospora uricase to homoge- repressed; serine and certain otheramino acids
neity and found that the protein appears to be are more active asrepressors than is ammonia,
a tetramer composed ofsubunits having a mo- suggesting that ammonia itselfmust be metab-
lecularweight ofapproximately 33,000. Uricase olized to an amino acid or other metabolite to
isastableenzymeandisnotsubjecttofeedback cause repression (13). The fact that oxalurate,
inhibitionbyammonia,glutamate,orglutamine. the gratuitous inducer, is transported into the
Reinert and Marzluf (78) studied the in vivo cells by a constitutive, energy-dependent per- D
stability of several of the purine catabolic en- mease is instructive, since it implies that the o
zymes; both uricase and allantoicase are stable failureofoxaluratetoinduceallophanatehydro- wn
enzymes, whereas allantoinase is quite labile lase during nitrogen repression does not result lo
bothinvivoandinvitro,withahalf-lifeinvivo from inducer exclusion. Apparently induction a
ofapproximately 20min. Itisnotclearwhether and repression are independent, and both di- de
allantoinase turnover has physiological signifi- rectly affect gene expression. G. Chisholm and d
cance,e.g.,bycontributingtothecontrolofflow T. G. Cooper (unpublished data) have recently fr
along this pathway. Allantoinase appears to be isolated mutants which define a new control om
extremelysensitivetoendogenousproteases,al- gene, dur5; the dur5 mutants produce the five

though it may be purified by using steps to activities required for allantoin catabolism in ht
quicklyseparate andprotectitfromproteases. highconstitutivelevels.Furthernore,durldur5 tp
Superimposeduponthemorefamiliarpattern double mutants, which cannot form any allo- ://
of nitrogen control of enzymes and permeases phanate, the inducer of the pathway enzymes, m
may be interesting developmental regulation. arestillconstitutivefortheremainingactivities, m
An instance occurs in Neurospora, in which implying that no type of internal induction is br
freshlyharvested conidia possess a generalsys- taking place. Thus, the dur5 gene apparently .a
temforpurinetranport;upongermination,this encodesarepressoractiveinnegativecontrolto sm
systemincreases and asecond, adenine-specific stop gene expression unless inducer is present. .
permease also develops (68). It is obviously of Mutants in dur5 are recessive to wild type, as or
great interest to understand such developmen- expected for a repressor gene. It should be em- g
/
tal-stage-specific regulation and how it may in- phasizedthatthisnegativecontrolbydur5iover o
teractwiththeusualnitrogen control ignals. these yeast catabolic enzymes is in sharp con- n

Allantoin canserve asanitrogensource inS. trast to the well-documented positive control A
cerevisiae, and its catabolism requires five en- (byuaY+) oftherelated genesinAspergillus. pr
zymes. The pertinent structural genes are lo- Yeast allantoin permease, at least one com- il 1
cated in two unlinked clusters; the final two ponent of which is coded for by dal4, is con- 0
enzymes, urea carboxylase and allophanate hy- trolledquitedifferentlythanaretheotherpath- ,
2
drolase, were thought to be encoded by contig- waycomponentsandisnotinducedbyallopha- 0
uous genes (durl and dur2) ofone cluster and nate (92); rather, allantoin itself, as well as two 19
to comprise a multienzyme complex (55). R. allantoinanalogs,inducetheallantoinpermease.
b
Sumrada, C. Lan, and T. G. Cooper (unpub- Allantoinin themedium can enter cellsbyway y
lished data) have recently found that the durl of a basal level of allantoin transport activity g
anddur2genesactuallyrepresentasinglestruc- thatisnormallypresent, andonce inside, itcan ue
tural gene whose product is a bifunctional pro- induce the allantoin pernease to full capacity s
tein(molecularweightof202,000) thatpossesses (45). Allantoin permease synthesis is also re- t
both urea carboxylase and allophanate pressed by nitrogen catabolite repression. Fi-
hydrolase activity (probably in two different nally, traninhibition ofallantoin permease ac-
globular domains). The second cluster is com- tivity is exerted by intracellular asparagine, as-
posed ofthree adjacent genes, dall, dal4, and partic acid, and lysine; this type of feedback
dal2, which encode allantoinase, allantoin per- inhibitionmayplayaregulatoryrolebyexclud-
mease, andallantoicase, respectively (21); these ingallantoinfromthecellwhenpreferablenitro-
enzymes exist as discrete polypeptides. More- gen sources are available (92). Turoscy and
over, the fact that the central gene, dal4, is Cooper(96) havealsoexaminedthetransportof
regulated differently than the outside genes allantoic acid in yeasts, which occurs by a con-
clearlyindicatesthattheyareexpressed assep- stitutive, energy-dependent system, distinct
arate units. fromtheallantoinperneasesystem. Duringthe
Allophanate,thelastinternediateofthepath- course of this work, the allantoinase reaction
446 MARZLUF MICROBIOL. REV.
was discovered to be readily reversible, but by protein synthesis began at about 10 min, and
using dall dal2 double mutants (lacking allan- active enzyme appearedat 12 min (12). Thus, a
toinase and allantoicase), transported allantoic major part ofthe lag period, about 6 min, ap-
acid accumulated without change. No efflux of peared to occur as a delay between the time
allantoate from preloaded cells could be de- transcriptionoccurredandtheenzymesynthesis
tected, which maybe dueatleastinparttothe began. One simple yet interesting conclusion is
sequestering ofintracellularallantoate inavac- that inducer uptake and all nuclear events re-
uole. quiredforturningongeneexpressionoccurvery
Lawtherand Cooper (54) studiedthekinetics rapidly, evenat220C.
of induction of allophanic hydrolase and ob- Cooper and his colleagues have also studied
served that enzyme activity increased within 3 thefunctionalhalf-life ofallophanate hydrolase
to4 minwheninducer (allophanate) wasadded mRNA and several other yeast mRNA's, mea- D
to cells growing at 360C. Its specific mRNA, suredascapacityforsubsequentenzymeorpro- o
w
measured asthe capacity for enzyme synthesis, tein synthesis (22). The observed mRNA half-
n
alsodecayedveryrapidly,withahalf-lifeofonly life wasfound to be strongly influenced by var- lo
3 min. Other yeast enzymes required about 10 ious experimental variables; e.g., mRNA turn- a
minforinduction, whichsuggestedthepossibil- over was increased at higher temperatures, d
e
itythatthatallophanichydrolase andtheother whereas inhibition of protein synthesis could d
three related enzymes might be controlled at a increase mRNA stability dramatically, in some f
r
post-transcriptional step. To investigate this cases up to 15-fold. Changes in nitrogen source o
m
possibility, lomofungin and cycloheximide were didnotinfluencethefunctionalhalf-livesofany

used to specifically interfere with transcription ofthe mRNA's examined. Clearly, comparison h
t
and translation, respectively. The results ofmessengerstabilityismeaningfulonlyifmea- tp
showed that: (i) induction occurredatthetran- surements are conducted under highly con- :/
/
scriptionallevel andrequired mRNA synthesis; trolledconditions.Inasinglecomparativestudy, m
(ii) synthesis of hydrolase mRNA began im- Lawther and Cooper (54) found that the half- m
mediatelyuponinductionandcouldoccurinthe livesofmRNA'sforinvertase,a-glucosidase,and b
r
absence of protein synthesis; (iii) the mRNA gross protein synthesis were all about 20 min, .a
half-life was increased when protein synthesis comparedwith3 minforallophanate hydrolase s
m
was blocked; and (iv) the enzyme itselfwasnot mRNA. The tentative conclusion from these
.
degraded if inducer was removed. It was con- resultsisthat, atleastinyeasts,mRNA'swhich o
r
cluded that both induction and repression of encode regulated enzymes may, as a class, turn g
/
allophanatehydrolasesynthesisoccurredatthe overconsiderablyfasterthandomRNA'swhich o
leveloftranscription,althoughinducermayalso specify constitutive enzymes. Obviously, mea- n

influencetherate ofmRNAtranslation (54). surementofanumberofmRNA'srepresentative A
Thesequenceofmoleculareventsinvolvedin of each class will be necessary to judge the pr
theinductionofallophanatehydrolasehasbeen correctness ofthishypothesis. il
1
studied byworkingwithyeast culturesat220C, 0
because at this temperature a 12-min lag oc- PROLINE CATABOLISM ,
2
curred before enzyme activity increased (12). It 0
was clear from prior results that induction re- Enzymesfortheutilizationofcompoundsthat 1
9
quired both RNA and protein synthesis, and it contain carbon but not nitrogen (e.g., acetate)
b
was ofinterest to determine what time periods are controlled simply by carbon catabolite y
were devotedtoeachduringthelagphase. Two repression. Similarly, enzymes involved in the g
yeast mutants were helpful since they interfere metabolismofcompoundscontainingjustnitro- u
e
withspecificaspectsofmacromolecularsynthe- gen(e.g.,nitrate) arecontrolledonlybynitrogen s
sis; rnal is a temperature-sensitive mutant be- catabolite repression. However, the metabolism t
lievedtobedefectiveintheprocessingortrans- ofcompoundsthatcanbeutilizedasbothnitro-
port from the nucleus of mRNA and other gen and carbon sources may be subject to both
RNAs. The prtl mutant is also temperature nitrogenandcarbonregulation.Thus,thestruc-
sensitive andisdefective ininitiationofprotein tural genes encoding certain enzymes may .be
synthesis at the restrictive temperature. Since subjecttomultipleregulatorysignals,whichim-
noyeastmutantsareyetavailablewhichspecif- plies that a complex control region containing
ically block initiation ofmnRNA synthesis, lom- two or more recognition sites may be situated
ofunginwasusedtoinhibitthisstep.Theresults nextto them.
ofthisseriesofexperimentsindicatedthatupon A gene cluster (in Aspergillus) comprised of
induction,mRNAsynthesiswasinitiatedwithin onepathway-specific regulatory gene andthree
1 to 1.5 min after inducer was added, nuclear structural genes which specifyproline catabolic
secretion ofthe mRNA occurred within 4 min, enzymes is apparently interrupted by a central